During the past several years this laboratory has concentrated on studies of the drug-metabolizing enzyme, the flavin-containing monooxygenase (FMO). Thought to comprise two forms at the most, we have discovered that the FMO is a gene family with at least five. Recently, we have begun to express these enzymes in COS-1, yeast, and E. coli systems in order to examine their substrate specificities and other properties. The properties of two of the enzymes, formerly called the "liver" and "lung" forms, in expression systems are the same as those observed with purified preparations with the exception of their kinetic behavior which is much less complex for the expressed enzymes. A third form that has been expressed exhibits unique substrate specificity in that is appears to metabolize only primary amines. However, neither hydrogen peroxide nor hydroxylamine equivalents are formed by the reaction. It is suspected that the hydroxylamine formed is further metabolized to the oxime. One notable common property among the sequences of the five FMOs is the inclusion of two putative pyrophosphate binding domains, one near the N-terminus and the second near residue 190. These areas, which account for the binding of FAD and NADPH to the enzyme are proposed to be critical for catalytic activity. We have modified the first of these sites by changing the GxGxxG sequence into GxGxxV and expressed the altered protein. This protein is devoid of activity and does not appear to have any FAD bound to it. A second notable common property among the five forms of FMO is the presence of an extremely hydrophobic C- terminal peptide comprising the final 26 amino acids. It has been proposed that the enzyme is anchored to the membrane via this peptide. We have examined the properties of the FMO expression product missing the C- terminal 26 residues. Results of this work demonstrate conclusively that the C-terminal hydrophobic peptide does not anchor the enzyme to the membrane. Future work will concentrate on the development of antibody preparations for each form of the FMO and on the elucidation of the physiological roles of these enzymes.